Novel Mitochondrial Ion Transporters

描述(申请人提供)：跨线粒体内膜的质子运输为ATP合成提供质子动力，但近年来，其他离子(如K+、Na+、钙离子和阴离子)的运输在生物能量学调节中的重要性日益明显。在过去的几年里，我们的重点主要集中在三个方面，i)表征参与保护缺血损伤的K+吸收途径，ii)了解Na+和Ca~(2+)动力学如何影响能量供需平衡，以及iii)表征内膜氧化剂敏感的能量耗散通道是如何被应激激活的。虽然在分离的线粒体、细胞和完整的心脏中，通过药理学工具和离子梯度的操纵已经学到了很多东西，但一个主要的限制是缺乏关于介导内膜离子运输的蛋白质的分子信息。基于详尽的蛋白质纯化和分级策略，以及在前一个资金阶段采取的旨在全面描述线粒体蛋白质组及其在缺血再灌注期间的修饰的团队方法，我们已经成功地确定了一些新的、高度可信的候选基因，我们认为这些候选基因是线粒体内膜上重要的K+、Na+和Ca~(2+)运输途径的基础。有趣的是，我们还发现了新的线粒体阴离子转运体，它们可能被证明参与了线粒体功能的调节。该项目将结合分子技术来操纵通过质谱学鉴定的线粒体蛋白质的表达水平，并在分离的线粒体和细胞中进行功能分析，以将特定的离子运输途径与其相应的蛋白质联系起来。基于已经通过质谱学获得的证据，我们相信我们正在明确地解决由线粒体ATP敏感(MitoKATP)和钙激活(MitoKCa)钾通道的成孔亚单位组成的分子实体，目前正在寻找可能介导线粒体钠钙交换(MNCE)和单价阳离子-氢交换(KHE或NHE)的强有力的候选分子。我们还将研究已识别的但未确定的阴离子转运体的可能功能作用，这些转运体可能参与线粒体体积调节和对氧化应激的反应。该项目的最终目标是克服线粒体生物学领域取得进展的一个重要障碍--对正常功能和缺血-再灌注病理生理学至关重要的离子转运蛋白的分子鉴定。

DESCRIPTION (provided by applicant): Proton transport across the mitochondrial inner membrane provides the protonmotive force for ATP synthesis, but the importance of the transport of other ions (e.g. K+, Na+, Ca2+, and anions) in the regulation of bioenergetics has become increasingly evident in recent years. Over the past several years, our focus has been on three main areas, i) characterizing the K+ uptake pathways involved in protection against ischemic damage, ii) understanding how Na+ and Ca2+ dynamics impact energy supply and demand balance, and iii) characterizing how inner membrane oxidant-sensitive energy dissipating channels are activated by stress. While much has been learned using pharmacological tools and manipulation of ion gradients in isolated mitochondria, cells, and intact hearts, a major limitation has been the lack of molecular information about the proteins mediating ion transport in the inner membrane. Based on an exhaustive protein purification and fractionation strategy, and the team approach undertaken during the prior funding period designed to fully characterize the mitochondrial proteome and its modifications during ischemia- reperfusion, we have been successful in identifying a number of novel, high confidence candidates that we believe underlie important K+, Na+ and Ca2+ transport pathways in the mitochondrial inner membrane. Intriguingly, we have also identified novel mitochondrial anion transporters that could prove to be involved in the regulation of mitochondrial function. This project will combine molecular techniques for manipulating the expression levels of mitochondrial proteins identified by mass spectrometry with functional assays in isolated mitochondria and cells to correlate a particular ion transport pathway with its corresponding protein. Based on evidence already obtained by mass spectrometry, we believe we are on track to unequivocally resolve the molecular entities comprising the pore forming subunits of the mitochondrial ATP-sensitive (mitoKATP) and calcium-activated (mitoKCa) potassium channels, and are currently pursuing strong candidates that could mediate mitochondrial sodium calcium exchange (mNCE) and monovalent cation-hydrogen exchange (KHE or NHE). We will also investigate the possible functional role of identified, but uncharacterized, anion transporters that may be involved in mitochondrial volume regulation and the response to oxidative stress. The ultimate goal of the project is to overcome a significant roadblock to progress in the area of mitochondrial biology - the molecular identification of ion transport proteins that are critical to normal function and to the pathophysiology of ischemia-reperfusion.